Publication Date


Document Type


First Advisor

Erman, James E.

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry


Ligand binding (Biochemistry); Hemoproteins; Nuclear magnetic resonance spectroscopy


The binding of small molecules to proteins can be used to probe the structure and chemical reactivity of the proteins' active sites. In this dissertation, the binding of imidazole and 4-nitroimidazole to the heme proteins, metmyoglobin and FixL, was studied. Stopped-flow kinetics studies suggest that metmyoglobin binds the imidazolate forms of these ligands faster than the imidazolium and neutral forms; however, spectroscopic data indicate that the neutral form is stabilized by metmyoglobin, even at high pH. Nuclear magnetic resonance experiments were used to find evidence that the neutral form of 4-nitroimidazole was bound at alkaline pH. There is evidence from these studies for a buried exchangeable proton, which is most logically assigned to the NδH proton of 4-nitroimidazole. If this assignment is incorrect, it does not rule out the presence of the proton, which could be buried in the diamagnetic envelope or in rapid exchange with water. Assignments for 21 out of 25 of the paramagnetically shifted resonances were also made. The FixL protein belongs to a class of heme proteins that functions as biological sensors. Ligand binding to FixL is not well characterized. The pH dependence of imidazole and 4-nitroimidazole binding to the FixL heme domain were used to investigate whether FixL discriminates between the differently charged forms of these ligands. The absorbance spectra of FixL complexes with imidazole and 4-nitroimidazole indicate that the neutral form of the ligands is stabilized at neutral and alkaline pH. The kinetic studies of imidazole binding to FixL show that the observed rate constant displays hyperbolic dependence on imidazole concentration. Therefore, the rate-limiting step at high imidazole concentrations must be associated with a change that occurs in the protein, such as a conformational change. An ionization on FixL increases the rate of this step. The heme 7-propionic acid carboxlyate group is the most likely candidate for this ionization. Estimates of the association rate constant, ka, for the heme domain of FixL, FixLN, indicate that imidazole and 4-nitroimidazole bind 185 times and 16 times faster to FixLN than metMb, respectively. The dissociation rate constant, kd, is also faster for FixLN than for metMb, resulting in FixLN equilibrium constants which are 15 times smaller and 2 times larger for imidazole and 4-nitroimidazole, respectively. The chemical reactivity at the active sites of these two oxygen-binding proteins is similar. Both metmyoglobin and FixL appear to bind the anionic forms of the ligands preferentially; however, both protein-ligand complexes must take up a proton upon ligand binding since the neutral forms of the ligands are bound, even at high pH.


Includes bibliographical references (pages [246]-249).


xiii, 253 pages




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